Resolution of Acanthamoeba castellanii chromosomes by pulsed field gel electrophoresis and construction of the initial linkage map

Pulsed field gel electrophoresis has been used to resolve chromosome-sized DNA molecules in fungi and parasites but has not yet been used successfully to examine the chromosomes of other lower eukaryotes used extensively for biochemical research such as Acanthamoeba, Physarum, and Dictyostelium. Here we show an electrophoretic karyotype of the protozoan Acanthamoeba castellanii using orthogonal field alternating gel electrophoresis (OFAGE). There are about 20 small chromosomes ranging in size from 220 kb to >2 Mb. We have assembled initial linkage groups assigning all of the cloned Acanthamoeba genes to chromosome-sized DNA molecules. Actin, suggested to have three or more non-allelic genes, maps to at least eight distinct chromosome bands. Two myosin II genes localize to two different chromosomal bands while myosin IB and 18S rRNA map to unresolved larger chromosomes.Abbreviations OFAGE Orthogonal field alternating gel electrophoresis     

Molecular detrapping and band narrowing with high frequency modulation of pulsed field electrophoresis.

In high electric fields, megabase DNA fragments are found to be trapped, i.e. to enter or migrate in the gel only very slowly, if at all, leading to very broad electrophoretic bands and loss of separation. As a consequence, low electric fields are usually used to separate these molecules by pulsed field electrophoretic methods. We report here that high-frequency pulses eliminate the molecular trapping found in continuous fields. When high frequency pulses are used to modulate the longer pulses used in pulsed field electrophoresis, narrower bands result, and higher fields can be used. We suggest that this is due to effects that occur on the length scale of a single pore.     

Mixed anionic detergent/aliphatic alcohol-polyacrylamide gel electrophoresis alters the separation of proteins relative to conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis

The order and relative mobility of proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is affected by unknown components that are differentially present in SDS preparations obtained from different sources [J.B. Swaney, G.F. Vande Woude, and H.L. Bachrach (1974) Anal. Biochem. 58, 337-346]. The modified separation capabilities of such SDS preparations are useful but the use of this phenomenon in a controlled manner requires that the components responsible for the altered separation be identified. Accordingly, this paper describes a polyacrylamide gel electrophoresis system [mixed alcohol/detergent-polyacrylamide gel electrophoresis (MAD-PAGE)] that employs a mixture of alcohol and detergent instead of SDS alone to modify and enhance protein separation relative to conventional SDS-PAGE. A defined mixture consisting of four sulfated alkyl detergents (dodecyl sulfate, tetradecyl sulfate, hexadecyl sulfate, octadecyl sulfate) as well as the four alcohols of corresponding aliphatic chain length was found to be effective at duplicating the electrophoretic effect of USP-grade SDS and thus changed the relative order and position of polypeptides on electrophoresis relative to conventional SDS-PAGE. This method serves as an adjunct to conventional SDS-PAGE by providing another means of resolving proteins that are not normally resolved by SDS-PAGE. Further, it was found that MAD-PAGE is capable of resolving the NS1 protein of influenza virus into three fractions, whereas conventional SDS-PAGE yields one electrophoretic species. Reelectrophoresis of these novel NS1 bands by conventional SDS-PAGE indicated that they were not modified during MAD-PAGE and probably represented distinct molecular forms present in infected cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophoretic mobility of lambda phage HIND III and HAE III DNA fragments in agarose gels: a detailed study

The electrophoretic mobility (μ) of DNA fragments from λ phage and ΦX 174, split by restriction enzyme to molecular lengths from 3 × 10² to 2.36 × 10⁴ base pairs, has been investigated in 0.6–4% agarose gels at various field strengths, ionic strengths, and temperatures. As already observed, μ is seen to be very sensitive to the field, increasing with field strength. The sensitivity increases with the molecular length of the DNA and decreases at high gel concentration. Our data are in qualitative agreement with recent theoretical predictions that concern the influence of the electric field on electrophoretic mobility. Mobility data have been extrapolated to zero field. This enables a comparison of our experimental results with theoretical predictions on the dependence of μ on the molecular weight of the DNA fragments. Our data fit, quite closely, a reptation model, where the tube path is described as a semiflexible entity with a persistence length equal to the pore diameter. The influence of the agarose concentration and the ionic strength of the buffer on the two parameters of the model—intrinsic electrophoretic mobility (μ₀) and the number of base pairs per element of the tube (g)—are well described by the model. The temperature dependence of the electrophoretic mobility, together with the influence of the agarose concentration on μ₀, indicate that the hydrodynamic drag is the leading frictional force on the DNA molecules in the gel.     

Influence of λ-DNA concentration on mobilities and dispersion coefficients during agarose gel electrophoresis

Earlier works demonstrated theoretically and experimentally that during gel electrophoresis the mobility μ and the dispersion coefficient D_x [reflecting band broadening; G. W. Slater and J. Noolandi (1985) Physics Review Letters, Vol. 55, pp. 1579–1582; T. A. Duke, A. N. Semenov, and J. L. Viovy (1992) Physics Review Letters, Vol. 69, pp. 3260–3263] depend on the chain length, the electric field, and on the gel concentration. Using a Fluorescence Recovery After Photobleaching setup coupled with an electrophoretic cell, we show that they also depend of the DNA concentrationC. Two regimes are observed. The first is analogous to a “dilute” regime in the gel where μ and D_x are DNA concentration independent. In the second regime, μ and D_x decrease when C increases. These results are explained by DNA-DNA interactions. As expected the C^* concentration, under which measurements must be carried out to avoid this effect, is found to be the same as the overlap concentration C^* determined in solution. Using concentrations of the studied DNA lower than its C^*, μ and D_x show a field dependence in good agreement with the predictions of the Biased Reptation model with Fluctuations. © 1997 John Wiley & Sons, Inc. Biopoly 42: 471–478, 1997     

Disc electrophoretic studies of hemoglobin dissociation by p-hydroxymercury benzoate

The reaction of excess p-mercurybenzoate with human hemoglobin produces five electrophoretic species on polyacrylamide gel. Only two of these bands have been previously observed when starch gel was employed. The chemical and electrophoretic studies presented in this paper illustrate that the appearance of an “extra” band in the β zone is due to the ability of PAGE to separate the βb₂^PMB ? β^PMB equilibrium to its discrete components. The remaining bands are accounted for by the superior resolution of PAGE over starch gel electrophoresis which allowed the detection of two (αβ)^PMB dimer species.     

Substrate-containing gel electrophoresis: sensitive detection of amylolytic, nucleolytic, and proteolytic enzymes

Electrophoresis of hydrolytic enzymes under nondenaturing conditions on acrylamide gels containing the appropriate high-molecular-weight substrates entrapped on the gel has been explored as a general method for sensitive enzyme resolution and detection. Under electrophoresis conditions of optimal enzyme activity, the enzymes may bind tightly to the fixed substrate and can only migrate in the electrophoretic field as the substrate is hydrolyzed. When the gels after electrophoresis in this “binding mode” are stained with substrate-detecting reagents, clear tracks of enzyme migration are observed, and the length of each track is a function of the amount of enzyme present in that track. Multiple forms of a given enzyme activity have not been and are not likely to be observed under these conditions. Under electrophoresis conditions of minimal (or suboptimal) enzyme activity, the enzymes do not bind to the fixed substrate and their mobility in the electrophoretic field does not appear to be significantly affected by the presence of substrate. After electrophoresis in this “nonbinding mode” the gels are incubated under conditions of optimal enzyme activity to allow substrate hydrolysis to take place before they are stained with substrate-detecting reagents, and active enzymes are detected as clear bands. Multiple forms of a given activity which were resolved during electrophoresis in the nonbinding mode are reflected by the presence of individual bands. The substrate-containing gel electrophoresis technique does not appear to be amenable to precise quantification of enzymes. By comparing the length of the clear tracks or the degree of staining of the activity bands for a range of enzyme concentrations, however, it is possible to establish the smallest amount of enzyme that can unequivocally be detected under a given set of conditions; from such studies we estimate that the sensitivity of detection with the substrate-containing gel electrophoresis technique can be orders of magnitude better than that obtained with other methods. The levels of detection observed in the work presented here were about 50 pg for α-amylase run on starch-containing gels, 1 pg to 1 ng for nucleases run on DNA- or RNA-containing gels, and 100 pg to 10 ng for 11 different pure and crude protease preparations run on gels containing heat-denatured bovine serum albumin.     

Analysis of the electrophoretic properties of double-stranded DNA and RNA in agarose gels at a finite voltage gradient

The electrophoretic mobilities of double-stranded (ds) DNAs and ds RNAs of various lenths, L, were measured in gels of 0.4–1.8% (w/v) agarose at a voltage gradient of 1.0 V/cm. Differences in the electrophoresis of ds DNA and ds RNA are presented and discussed. A general expression is derived that describes the electrophoretic mobility, M, of either type of ds nucleic acid as a function of the gel concentration and the nucleic acid length: M = M′₁(L/L₀)^?x ? M₂, where M′₁ and L₀ are constants, and x and M₂ depend on the agarose gel concentration. The results obtained by fitting our data with this equation are consistent with the mobilities of nucleic acids in a wide range of gel concentrations, including free electrophoresis in solution and electrophoresis in gles of high agarose concentration in which nuleic acids are expected to reptate through the gel matrix. Finally, various methods of plotting agarose gel electrophoresis data are discussed.     

Development of a dedicated two-dimensional gel electrophoresis system that provides optimal pattern reproducibility and polypeptide resolution.

The development of a dedicated two-dimensional gel electrophoresis system is described that provides superior performance in terms of high resolving power and enhanced gel-to-gel reproducibility. Isoelectric focusing is performed in a 1-mm capillary tube with a 0.08-mm thread, optimized for this application, incorporated along its length prior to polymerization of the gel matrix. The isoelectric focusing gel is 4% T, 2.6% C to minimize sieving of proteins and promote adhesion of the gel to the thread. The thread incorporated in the isoelectric focusing matrix prevents gel stretching and breakage during its application to the second dimension. An optimum ampholyte pH range has been defined based on 1600 polypeptides present in a transformed fibroblast cell lysate and verified using a variety of other cell types. The length of time required to complete an electrophoretic separation in the second dimension was found to depend on buffer conductivity establishing the importance of high quality electrophoresis grade reagents devoid of contaminating salts. To ensure reproducibility of electrophoretic separations, it is critical to maintain a strict control of temperature during the second dimension separation. This prevents altered migration of some polypeptides relative to neighboring polypeptides that have constant Rfs over a broad temperature range. It was also determined that to obtain the maximum information from a complex protein mixture it is critical to use a large format 22- x 22-cm two-dimensional electrophoretic system. Using the optimized two-dimensional electrophoretic system and computerized gel analysis, it was determined that molecular weight estimates of polypeptides differed by approximately 350 daltons between gels, while isoelectric point estimates differed by approximately 0.03 pH units between gels. Using the two-dimensional electrophoresis system described, approximately 1000 polypeptides can be routinely detected from silver-stained 10% polyacrylamide gels or 1600 polypeptides from autoradiographs of 35S-methionine-labeled polypeptides.     

Electrophoretic transfer protein zymography

Zymography detects and characterizes proteolytic enzymes by electrophoresis of protease-containing samples into a nonreducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel containing a copolymerized protein substrate. The usefulness of zymography for molecular weight determination and proteomic analysis is hampered by the fact that some proteases exhibit slower migration through a gel that contains substrate protein. This article introduces electrophoretic transfer protein zymography as one solution to this problem. In this technique, samples containing proteolytic enzymes are first resolved in nonreducing SDS–PAGE on a gel without protein substrate. The proteins in the resolving gel are then electrophoretically transferred to a receiving gel previously prepared with a copolymerized protein substrate. The receiving gel is then developed as a zymogram to visualize clear or lightly stained bands in a dark background. Band intensities are linearly related to the amount of protease, extending the usefulness of the technique so long as conditions for transfer and development of the zymogram are kept constant. Conditions of transfer, such as the pore sizes of resolving and receiving gels and the transfer time relative to the molecular weight of the protease, are explored.     

A discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide slab gel system of high resolution

A highly porous and efficient discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system is described. The slab get consists of two porous layers of acrylamide of the following composition: 4% acrylamide, 0.04% bisacrylamide for the stacking gel, and 10% acrylamide, 0.1% bisacrylamide for the separating gel, both layers having different buffers. The separating gel mixture (final pH 9.0) and the buffers of the electrode chamber (pH 8.45) consist of Tris and glycine in such a ratio that no acid or base is necessary to adjust the pH. The resulting gel system has the following advantages: (a) it is able to resolve the components from large-volume samples (up to 200 microliter) after an overnight electrophoresis run while still maintaining the capacity to produce very sharp bands; (b) it has a high and broad resolution, allowing the separation on the same gel of proteins with apparent molecular masses between 10,000 and 450,000 Da; (c) it is very easy to prepare and shows excellent reproducibility in the electrophoretic patterns; (d) when used as a second dimension in tandem with isoelectric focusing, it improves the resolving power of two-dimensional gel electrophoresis; and finally, (e) its low crosslinker-to-acrylamide ratio allows the effective and rapid transfer of proteins to nitrocellulose membrane, thus improving the usefulness of protein blotting. In all cases, adrenal medullary chromaffin cell proteins were used as test samples.     

Long range periodicities in mouse satellite DNA.

Escherichia coli restriction enzyme II breaks mouse satellite DNA into fragments which form a series of bands on gel electrophoresis. The DNA in the strongest band has a length of 220 to 260 nucleotide pairs and the other bands are multiples of this length. It is shown that these fragments are linked together in long arrays in the satellite sequence. The reassociation register of the DNA is about half the length of the 220 to 260 nucleotide pair fragment. In the electrophoresis pattern of the Eco RII² fragments other weaker bands can be seen. The stronger bands of the minor patterns fall half-way between the bands of the main pattern and the smallest is 120 to 130 nucleotide pairs long. The properties of the minor fragments suggest short spacings of the restriction site which have been produced by unequal crossing-over. The extents of divergence and unequal crossing-over are estimated. From this analysis and the sequence analysis described in the accompanying paper (Biro et al., 1975) it is proposed that mouse satellite DNA consists of an hierarchy of four periodicities which reflect stages in the evolution of the sequence.Digestion of mouse satellite DNA with Hae III produces fragments with the same sizes as those produced by Eco RII, but the yields are much lower. It is suggested that Hae III sites have been introduced by divergence and subsequently spread by unequal crossing-over.     

Separation of single-stranded ribonucleic acids by acrylamide-agarose-urea gel electrophoresis

The incorporation of 6 m urea into polyacrylamide-agarose gels reduces the width of the bands formed upon electrophoresis by single-stranded RNA molecules in the 10⁵- to 10^6.5-dalton size range. As a result the resolving power of gels is increased and molecules that differ in size by as little as 5% can be separated.     

Design and calibration of a semi-synthetic DNA phasing assay

Electrophoretic assays of intrinsic DNA shape and shape changes induced by ligand binding are extremely useful because of their convenience and simplicity. The development of calibrations and empirical quantitative relationships permits highly accurate measurement of DNA shape using electrophoresis. Many conventional analyses employ the unidirectional ligation of short DNA duplexes. However, many oligonucleotides (typically more than 20) must often be synthesized for a single experiment. Additionally, the length of the DNA duplex can become limiting, preventing the analysis of certain DNA sequences. We now describe a semi-synthetic electrophoretic phasing method that offers several advantages, including a reduced number of required synthetic oligonucleotides, the ability to analyze longer DNA duplexes and a simplified approach for data analysis. We characterize semi-synthetic DNA probes in electrophoretic phasing assays by ligation of synthetic duplexes containing A₅ tracts between two longer restriction fragments. Upon electrophoresis, the gel mobility is strongly correlated with the predicted DNA curvature provided by the reference A₅ tracts. Having obtained this calibration, we show that the semi-synthetic phasing assay can be readily and economically applied to analyze DNA curvature induced by DNA charge modifications and DNA bending due to peptide binding.     

Electrophoretic karyotype of Saprolegnia monoica

Abstract The electrophoretic karyotype of Saprolegnia monoica was determined by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Eight chromosomal bands were separated. The size of these bands, based on migration relative to those of chromosomal DNA of Saccharomyces cerevisiae , Schizosaccharomyces pombe and Hansenula wingei , is estimated to be between 0.9 and 5.8 Mb. The genome size is estimated to be 51 Mb.     

Comparison of the separation of Candida albicans chromosome-sized DNA by pulsed-field gel electrophoresis techniques.

Pulsed-field gel electrophoresis techniques were used to study chromosome-sized DNA molecules of C. albicans. Chromosome-sized DNA of two strains of Candida albicans has been resolved into 8 bands by orthogonal-field-alternation gel electrophoresis (OFAGE). Six bands were observed in chromosomal preparations of C. albicans using field-inversion gel electrophoresis (FIGE). Differences in the electrophoretic mobilities of bands of the strains of C. albicans examined suggests that chromosome-length polymorphisms exist and make it difficult to correlate the banding patterns among strains. These correlations were facilitated, however, by assignment of C. albicans chromosomes by hybridization using a collection of cloned DNA probes specific for each of the 8 observed bands. Southern blotting showed that the 6 FIGE bands consisted of 4 singlets and 2 comigrating doublets, accounting for the 8 bands observed by OFAGE analysis. The agreement between OFAGE and FIGE analysis suggests that the C. albicans haploid genome contains a minimum of 8 chromosomes.     

Separation of chromosomal DNA molecules from C.albicans by pulsed field gel electrophoresis.

Modifications have been made to standard pulse field gel electrophoresis (PFGE) systems to enable very large DNA molecules to be resolved. The single most important modification was to elevate the temperature of electrophoresis to 35 degrees C. This enabled the largest Saccharomyces cerevisiae chromosome to be reproducibly resolved. More impressively, it enabled the DNA of Candida albicans to be clearly resolved into six bands, a feat which was very difficult at lower temperatures. Even so, optimal resolution could only be obtained by carefully adjusting field voltages and switching times. The DNA from the two largest C. albicans chromosomes, which was estimated to be at least 5-10Mbp in size, ran somewhat anomalously, giving fuzzy bands which did not migrate in the direction of the average electric field. That the highest molecular weight band was a distinct chromosome was demonstrated by specific hybridisation to the C. albicans ADE2 gene probe. With further fine tuning, the PFGE system described here should be capable of resolving DNA from the smallest human chromosomes.     

Sequential two-dimensional and acetic acid/urea/Triton X-100 gel electrophoresis of proteins

A method is described which combines the resolving power of two-dimensional gel electrophoresis with that of acetic acid/urea/Triton X-100 gel electrophoresis, avoiding the necessity of eluting protein from the gels at any step of the procedure. The combination of electrophoretic separation on the basis of charge, mass, and hydrophobic properties of the proteins has the potential of resolving modified forms and isoforms present in very complex protein populations. The technique can be used for analytical purposes, or it may be scaled up to yield microgram amounts of highly purified proteins. The resolution obtained by tandem application of nonequilibrium pH gradient electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and polyacrylamide gel electrophoresis in the presence of nonionic detergent was evaluated using crude nuclear proteins of the nematode Caenorhabditis elegans.     

A small (58-nm) attached sphere perturbs the sieving of 40-80-kilobase DNA in 0.2-2.5% agarose gels: analysis of bacteriophage T7 capsid-DNA complexes by use of pulsed field electrophoresis.

Although the icosahedral bacteriophage T7 capsid has a diameter (58 nm) that is 234-fold smaller than the length of the linear, double-stranded T7 DNA, binding of a T7 capsid to T7 DNA is found here to have dramatic effects on the migration of the DNA during both pulsed field agarose gel electrophoresis (PFGE; the field inversion mode is used) and constant field agarose gel electrophoresis (CFGE). For these studies, capsid-DNA complexes were obtained by expelling DNA from mature bacteriophage T7; this procedure yields DNA with capsids bound at a variable position on the DNA. When subjected to CFGE at 2-6 V/cm in 0.20-2.5% agarose gels, capsid-DNA complexes arrest at the electrophoretic origin. Progressively lowering the electrical potential gradient to 0.5 V/cm results in migration; most complexes form a single band. The elevated electrical potential gradient (3 V/cm) induced arrest of capsid-DNA complexes is reversed when PFGE is used instead of CFGE. For some conditions of PFGE, the mobility of capsid-DNA complexes is a function of the position of the capsid on the DNA. During either CFGE (0.5 V/cm) or PFGE, capsid-DNA complexes increasingly separate from capsid-free DNA as the percentage of agarose increases. During these studies, capsid-DNA complexes are identified by electron microscopy of enzymatically-digested pieces of agarose gel; this is apparently the first successful electron microscopy of DNA from an agarose gel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Orientational dynamics of T2 DNA during agarose gel electrophoresis: influence of gel concentration and electric field strength

The understanding, on a molecular level, of the mechanisms responsible for the improved separation in DNA gel electrophoresis when using modulated electric fields requires detailed information about conformational distribution and dynamics in the DNA/gel system. The orientational order due to electrophoretic migration ("electrophoretic orientation") is an interesting piece of information in this context that can be obtained through linear dichroism spectroscopy [M. Jonsson, B. Akerman, and B. Nordén, (1988) Biopolymers 27, 381-414]. The technique permits measurement of the orientation factor S of DNA (S = 1 corresponds to perfect orientation) within an electrophoretic zone in the gel during the electrophoresis. It is reported that the degree of orientation of T2 DNA [170 kilo base pairs (kpb)] is considerable (S = 0.17 in 1% agarose at 10 V/cm) compared to relatively modest orientations of short fragments found earlier (for 23-kbp DNA, S = 0.03 in 1% agarose at 10 V/cm), showing that large DNA coils are substantially deformed during the migration. Growth and relaxation dynamics of the orientational order of the T2 DNA are also reported, as functions of gel concentration (0.3-2%), electric field strength (0-40 V/cm), and pulse characteristics. The rise profile of the DNA orientation, when applying a constant field, is a nonmonotonic function that displays a pronounced overshoot, followed by a minor undershoot, before it reaches steady-state orientation (after 12 s in 1% agarose, 9 V/cm). The orientational relaxation in absence of field shows a multiexponential decay in a time region of some 10 s, when most of the DNA anisotropy has disappeared. A surprising phenomenon is a memory over minutes of the DNA/gel system to previous pulses: with two consecutive rectangular pulses (of the same polarity), the orientational overshoot and undershoot as a response to the second pulse are significantly reduced compared to the first pulse. The time required to recover 90% of their amplitudes is typically 1200 s (1% agarose, 9 V/cm), which may be compared to the time required to relax 90% of the DNA orientation, which is only 6 s. The major part of the over- and undershoot recovery is thus a reorganization of a system in which DNA is already randomly oriented. The different response amplitudes and relaxation times, including the amplitude and recovery time of the overshoot, of the orientational order of DNA in the electrophoretic gel have been studied as functions of gel concentration and field strength. The results are discussed against relevant theories of polymer dynamics.